N-(mercaptoacyl)aminoacids

ABSTRACT

Compounds useful for prophyaxis and therapy in treating a metabolic disorder, such as, nosotoxicosis due to a heavy metal, radiation disorder, diabetes or hepatitis and represented by the formula   and intermediates therefor are disclosed.

United States Patent [1 1 Mita et a1.

1 1 N-(MERCAPTOACYL)AMINOACIDS 175] Inventors: Itaru Mita, Ashiya; Shigeo Okumura, Takarazuke; Shigeru Yamabe, Kobe; Yoshihiko Funae, Suita; Junzo Matumoto, Osaka, all

of Japan [73] Assignee: Santen Pharmaceutical Co. Ltd.,

Osaka, Japan [22] Filed: Sept. 20, 1973 211 App]. No.: 398,934

[30] Foreign Application Priority Data Oct. 3, 1972 Japan 47-10049] May 11, 1973 Japan 48-52924 \[52] US. Cl. 260/470; 260/455 R; 260/455 B;

260/481 R; 424/301; 424/309; 424/311 [51] Int. Cl. C07C 149/00 [58] Field of Search 260/516 1451 July 29,1975

156] References Cited UNITED STATES PATENTS 3,246,025 4/1966 Mlla et a1. 260/516 FOREIGN PATENTS OR APPLICATIONS 1,023,003 3/1966 ;United Kingdom Primary Examiner-John F. Terapane Attorney, Agent, or Firm-Flynn & Frishauf 57 ABSTRACT and intermediates therefor are disclosed.

6 Claims, N0 Drawings N-( MERCAPTOACYL )AMINOACIDS R-CH-CONHCH-(CHZ),,COOH

wherein R is a member selected from the group consisting of alkyl. aryl and aralkyl radicals; R is a member selected from the group consiting of a hydrogen atom an accelerating action of metabolism, but also are low in toxity and have a high transitivity into tissue. These compounds are. therefore. useful for prophylaxis and therapy in treating a metabolic disorder such as nosotoxicosis due to a heavy metal. radiation disorder. diabetes or hepatitis. This invention provides an industrially convenient process for preparing these useful compounds.

The transitivity into tissue and the radioresisting action as to each resisting action were indicated as percent survival of mice and partition coefficient. respectively. The percent survival of mice was determined by use of fluorescence X-ray as follows.

The known compound. 2-mercaptopropionylglycine was used in this test as control. The mice was divided into groups of mice each. The members of groups were administered with test compounds in the dose indicated. respectively. After the administration. the mice were irradiated with fluorescence X-ray to give the total dose of 800R as y-rays and 30 days after the irradiation. the number of surviving mice was counted. The test results are shown in the following table.

Table Percent of mice surviving 30 days after the y-ray Partition irradiation for coefficient the total dose Dose (Butanol/ Test Compounds of 800R (mg/kg) BuffenpH 7.4)

None 17.1 N-(Z-mercaptopropionyl) 43.0 (20) 0.17 glycine 31.0 (10) N-( Z-mercaptoisovaleryl) 20.0 10) 0.92 glycine N-(Z-mercaptophenylacetyl) 54.5 10) 1.03 glycine 31.5 (5) N-( Z-mercaptophenyl- 51.5 (10 2.54 propionyl)glycine 25.7 5) N-(Lmercapto-p- 1 1.4 10) 3.98 chlorphenylacetyl)glycine N-(2-mercaptopropionyl 48.0 10) 0.21 B-alanine N-( Z-mercaptopropionyU- 24.0 10) 1.88 leucine N-(Z-mercaptopropionyUS- 60.0 10) 0.63 aminocaproic acid N-(Lmercaptopropionyh- 46.0 (10) 1.73 phenylgl cine N-(2-me :aptopropionyl)- 52.0 (10) 3.68 phenylalanine H* N-(Z-mercaptopropionyl- 48.0 10) 3.55 pheny1)g1ycine L* *H and L indicate diasterecmers having a high melting point and a low melting point. respectively. which may be prepared in accordance with Example VI hereinbelow.

and alkyl. aryl and aralkyl radicals and n is an integer of from 0 to 4; when n is an integer of from 1 to 4, R and R being CH and H respectively; when n is zero, R being CH and R being a member selected from the group consisting of CH CH C H CH and (CHM- CHCH or R being a member selected from the group consisting of (CH;,) CH. C..H,-,. p-Cl-C H and C H CH and R being H.

Compounds of this invention have not been distiosed in any literature and are useful as therapeutic agents having various pharmacological activities. Tlamely, these compounds not only have an accelerating action for elimination of a heavy metal such as mercury. an eliminating action with respect to a substance harmful to human body such as a free radical or a peroxide and The compounds of this invention may be prepared by various known processes. but the following process is the most convenient because it gives the highest yield.

This process comprises reacting a compound reprein a solvent in the presence of an alkaline agent to form a compound represented by the formula reacting the compound with a salt of thiohenzoic acid or xanthogenic acid to form a compound represented by the formula and subjecting the compound to hydrolysis. ln the above formulae, R. R and n are as defined above: X and X are independently a halogen atom: and M is a thiobenzoic acid or ethylxanthogenic acid residue.

Examples of the alkaline agent are hydroxides. carbonates of sodium and potassium and ter.-amine salt such as pyridine and triethylamine salts and the like. The reaction of the halide with the aminoacid in the presence of an alkali is conveniently carried out in a solvent such as water. ether, benzene. chloroform, acetone, dioxane at a low temperature while maintaining the pH of the reaction mixture at neutral or slight alkalinity.

The resulting compound represented by the formula was optionally isolated and reacted with a salt of thiobenzoic acid or alkylxanthogenie acid in water or a proper organic solvent, for example. a lower alcohol or ethyl acetate at a low temperature to give an S- substituted compound. This compound may be subjected to hydrolysis with the acid of a hydroxide of alkali metal, alkaline earth metal or ammonia in water or in a lower alcohol at room temperature or an elevated temperature to disconnect the substituent. The N-(Z- mercaptoacyl) aminoacid thus obtained may be readily purified by recrystallization from an organic solvent such as ethyl acetate.

The present invention is further illustrated by the following examples. but they are not to be construed as limiting the present invention.

EXAMPLE I 200 ml of thionylchloride was added to 62.9g (0.293 mol) of a-bromophenylacetic acid and the resulting mixture was refluxed for 2 hours. After the completion of the reaction. excess thionylchloride was removed from the reaction mixture under reduced pressure and then the reaction mixture was distilled to yield 618g of Z-bromophenylacetic chloride (b.p. 105C/5 mmHg).

Then, 7.5g (0. l mol) of glycine was neutralized with 50 ml of ZN-NaOH and to the resulting solution were added dropwise over one hour 20.0g (0.085 mol) of 2- bromophenylacetylchloride and 43 ml of ZN-NaOH while stirring. During the addition, the reaction mixture was maintained at a temperature below 5C and adjusted to a pH of about 8. After the completion of the addition. the mixture was stirred at room temperature for 4 hours to complete the reaction. To the resulting solution. without isolating a bromine-containing compound. was added the filtrate which had been prepared by neutralizing 13.8g (0.10 mol) of thiobenzoic acid with ml of ZN-KOH and the thus obtained solution was filtrated to remove undissolved substances. and then the mixture was allowed to stand overnight at room temperature while adjusting the pH to about 8. On the next morning. the solution was acidified with hydrochloric acid while ice-cooling to precipitate crystals. The cyrstals were recovered by filtration. washed with water. dried and recrystallized from benzene to give 23.9g of 2-benzoylmercaptophenylacetylglycine as white crystals (m.p. l33-l38().

9.9g (0.03 mol) of they Z-benzoylmercaptophenylacetylglycinc was added to 50 ml of concentrated aqueous ammonia and the mixture was stirred at room temperature for one hour. During stirring and about 30 minutes after the addition. the formation of benzamide was observed. After the completion of the reaction. the benzamide was removed by extraction with ether. After removing the ammonia under reduced pressure. the aqueous layer was acidified with hydrochloric acid and extracted with ethyl acetate. The extract was washed with water and dried over anhydrous sodium sulfate. After the ethyl acetate had been distilled off. a small amount of benzene was added to the extract to give N-(Z-mercaptophenylacetyl)glycine. C H -,CH(SH)CONHCH- COOH. as white crystals. Yield: 5.6g, (m.p. 91 93C).

Infrared Spectrum EXAMPLE [I 2.9g of the bromine-containing compound prepared by the method according to Example I was neutralized with an alkali and then 2.4g (1.5 times mol as great as that of the compound) of potassium ethylxanthogenate was added to the neutralized compound followed by stirring at room temperature overnight. The next morning, the reaction mixture was acidified witih hydrochloric acid to give 3.3g of N-(2-ethylxanthogenphenylacetyl )glycine.

as white crystals. (m.p. 133 l35.5C)

Infrared Spectrum 1055 cm (xanthate);

By the hydrolysis 'of the thus obtained xanthate. N- (Z-mercaptophenylacetyl)glycine mentioned in Example I could readily be obtained.

EXAMPLE Ill 605g of Z-bromo-3-phenylpropionic acid was mixed with 100 ml of thionylchloride and the mixture was refluxed for 3 hours and. after the excess thionylchloride had been distilled off, was distilled under reduced pressure to give 46.2g of 2-bromo-3-phenylpr0pionylchl0- ride. (b.p. 100 101C/4 mmHg) g of 2-bromo-3'-phenylpropionylchloride was added dropwise to the solution which had been prepared by dissolving Zl.'8g1of glycine and l9.4g of NaOH in 250 ml of water over 1 hour while stirring and ice-cooling. After completion of the addition. the mixture was stirred at room temperature for additional 4 hours. The mixture was maintained at a pH of 8 during the reaction. After completion of the reaction. the reaction mixture was acidified with hydrochloric acid to form 70.9g of 2-bromo-3-phenylpropionylglycine having a melting point of 148C after recrystallization from ethyl acetate.

The thus formed bromine-containing compound was reacted with 43.2g of thiobenzoic acid in a similar way in accordance with Example 1 to give 85.0g of 2- benzoylmercapto-3-phenylpropionylglycine which had a melting point of 181 182C after recrystallization from ethylacetate-ethanol.

The thus obtained compound was hydrolized in a concentrated aqueous ammonia solution in a manner similar to that used in Example 1 to give N-(2 mercapto-3-phenyl)glycine. C..H,-,CH- ,CH(SH)CONHCH COOH. (m.p. 122 125C) at a yield of 81%. After recrystallization from ethyl acetate, the compound had a melting point of 126 127C and was 100% pure as determined by iodometry.

lnfrared Spectrum 3260 cm" (NH); 1720 cm", 1705 cm (COOH); 1635 cm", 1560 cm"(CONH) When L2'bromo-3-pheny1propionic acid was used as a starting material. optically active N-(Z-mercapto- 3-phenylpropiony1)glycine was obtained. (m.p. 55 58C. [01],, 5.3).

lnfrared Spectrum 3260 cm (NH); 1710 cm (-COOH); 1635 cm. 1550 cm (CONH-) EXAMPLE 1V 28.3g (0.1 13 mol) of 2-bromo-p-chlorophenylacetic acid was mixed with 26.9g (0.226 mol) of thionylchloride and the mixture was refluxed for 2.5 hours. After the excess thionylchloride had been distilled off, the reaction mixture was distilled under reduced pressure to give 26.7g of 2-bromo-p-chlorophenylacetyl chloride as yel1oworange oil. (b.p. 148 152C/l5 mmHg) 4.9g (0.065 mol) of glycine was dissolved in 250 ml of 0.5 NNaOH and then to the solution was added dropwise 14.6g (0.054 mol) of Z-bromo-pchlorophenylacetyl chloride over 1 hour while stirring and cooling with a freezing mixture. After completion of the addition. the reaction mixture was stirred at room temperature for an additional 2 hours. During stirring. the pH of the mixture was adjusted to 8. The filtrate which had been prepared by dissolving 11.6g (0.08mol) of thiobenzoic acid in 40 ml of 2NKOH and filtering the solution to remove undissolved substances was added to the reaction mixture followed by stirring at room temperature overnight. The next morning. the mixture was acidified with hydrochloric acid while icecooling to precipitate crystals. These crystals were recovered by filtration. washing with water and washing with benzene to give 151g of crude N-(Z- benzoylmercapto-p-chlorophenylacetyl)glycine. The product had a melting point of 158 159C after recrystallization from ethyl acetate-benzene.

1 1.0g (0.03 mol) of N-(Z-benzoylmercapto-pchlorophenylacetyl)glycine was dissolved in 75 ml of concentrated aqueous ammonia followed by stirring at lnfraredSpectrum 3260 cm (NH): 1760 cm (--COOH): 1610 cm. 1540 cm (--CONH) EXAMPLE V 50g of Z-bromoisovaleric acid was mixed with ml of thionylchloride and the mixture was refluxed for 4 hours to give 45.9g of 2-bromoisovaleryl chloride. (lxp. 68 70C/29 mmHg) 501g of the resulting valerylchloride and 10g of triethylamine were added dropwise over 1 hour to the solution, which had been prepared by neutralizing 121.21g (0.25 mol) of glycine with 10.0g ofNaOH in ml of water. while stirring and ice-cooling and followed by stirring at room temperature for an additional one hour. Then the mixture was acidified with hydrochloric acid to give 51.6g of 2-bromoisovalerylglycinc. (m.p. 138 139C).

In a manner similar to that used in any one of the foregoing Examples 27.5g (0.116 mol) of 2- bromoisovalerylchloride was reacted with 20.6g (0.15 mol) of thiobenzoic acid by stirring at room temperature for 40 hours. After completion of the reaction. the reaction mixture was acidified with hydrochloric acid to give 290g of 2 benzoylmercaptoisovalerylglycine having a melting point of 136 137C after recrystallization from ethyl acetate.

lnfrared Spectrum 3360 cm' (NH); 1735. 1215. 920 cm" (benzoate); 1650 cm (COOH); 1615. 1540 cm (CONH) In a manner similar to that used in Example 1. N-(2- benzoylmercaptoisovaleryl)glycine was hydrolized with concentrated aqueous ammonia to give N-( 2-mercaptoisovaleryl )glycine. (CH;,) CHCH( SH )CONHCH- COOl-l having a melting point of 1 18 119C after recrystallization from ethyl acetatebenzene.

lnfrared Spectrum 3280 cm(NH-); 2500 cm(-SH); 1750 cm"' (COOH); 1605. 1560 cm '(-CONH) EXAMPLE Vl 33g (0.20 mol) of DL-phenylalanine was neutralized with 100 ml of 2N-NaOH solution and to the resulting solution were simultaneously added dropwise 34.2g (0.20mol) of 2-bromo-propionylchloride and 1 10 ml of 2N-NaOH solution over about 1 hour while stirring and ice-cooling. During the reaction. the reaction mixture was maintained in a slightly alkaline condition. After completion of the addition, the ice-bath was removed and the mixture was stirred at room temperature for a further 2 hours. The filtrate which had been prepared by neutalizing 33.7g (0.24 mol) of thiobenzoic acid with 122 ml of ZN-KOH solution and filtering the thus obtained solution was added to the reaction mixture. The mixture was stirred at room temperature overnight and then acidified with hydrochloric acid while icecooling to form oil. The oil was extracted with ethyl acetate and the extract was washed with water. dried over anhydrous sodium sulfate. and then allowed to be placed under reduced pressure to remove the ethyl acetate. During removal of the ethyl acetate. crystals started to precipitate. After completion of the evaporation. benzene was added to these crystals and they were recovered by filtration and washed with benzene to give 250g of N-( 2'benzoylmercaptopropionyl phenylalanine. (m.p. l73- 175C) Infrared Spectrum CH CHCONHCHCOOH.

SH CH-g u n The compound of a higher melting point (m.p. 131

Infrared Spectrum 3320 Cm '(Nl-I); 1715 cm"(COOH); 1620.

1525 cm (-C()NH) Analysis Found: C.57.00; H. 6.01; N. 5.54 Calcd.: C. 56.81; H. 5.97; N. 5.33

The compound having a lower melting point (m.p. 105 108C) Infrared Spectrum Analysis Found: C. 57.17; H. 6.06; N. 5.54 Calcd: C. 56.81;

EXAMPLE VII 13.1g (0.10 mol) of DL-leucine was neutralized with 50 ml of 2N-NaOH solution and to the solution were simultaneously added dropwise 17.1g (0.10 mol) of 2-bromopropionylchloride and 50 ml of ZN-NaOH solution at room temperature over one hour while stirring and maintaining the reaction mixture in a slightly alkaline condition. After completion of the addition, the ice bath used was removed and the mixture was stirred at room temperature for 3 hours. successively, to the mixture was added the filtrate which had been prepared by neutralizing 138g (0.10 mol) of thiobenzoic acid with 50 ml of ZN-KOH solution and filtering. The resulting mixture was stirred at room temperature overnight and then acidified with hydrochloric acid to form oil. After extraction of oil with ethyl acetate. the oil was purified by subjecting it to chromatography using a silica gel column to give 185g of N-(2-benzoylmercaptopropionyllleucine as oil. 40 ml of concentrated aqueous ammonia was added to 6.0g of 2-benzoylmercaptopropionylleucine to allow the derivative of leucine to hydrolyze in a manner similar to that used in Example I to give 2.6g of N-(2-mercaptopropionyl)leucine.

(m.p. 77 84C) Analysis Found: C. 49.54; H. 7.94; N. 6.68 Calcd.: C. 49.30: H. 7.82; N. 6.39

EXAMPLE VIII 8.9g (0.10 mol) ofB-alanine was dissolved in ml of water. To the solution was added 20 mlof triethylamine and then was added dropwise 22.2g (0.13 mol) of 2hromopropionylchloride over 1 hour while stirring and ice-cooling. The mixture was stirred for a further 2 hours. To the mixture was added the filtrate which had been prepared by neutralizing 19.5g (0.14 mol) of thiobenzoic acid with an aqueous KOH and filtering the solution. followed by stirring overnight. Crystals pre cipitated were recovered by filtration. washed with water and dried to give 20.8g of N-(2- benzoylmercaptopropionyl)B-alanine. After recrystallization from benzene. the product had a melting point of 94 97C.

Infrared Spectrum The product had a melting point of 100 103C after recrystallization from ethyl acetate.

Infrared Spectrum 3250 cm Nl-I); 1700 cm (COOH); 1635 cm, 1560 cm (CONH) Analysis Found: C. 40.50; H. 6.29; N, 7.96 Calcd.: C. 40.68; H. 6.26; N. 7.91

EXAMPLE IX 50g (0.383 mo l) of S-aminocaproic acid was added to 200 ml of water and 10 ml of pyridine was added to Infrared Spectrum 3280 cm"(NH); 1660 cm. 1530 cm (CONH); 1720 cm '(COOH); 910 cm"(S- Benzoate) 124g of N-( 2-benzoylmercaptopropionyl )5- aminocaproic acid was hydrolized with NaOH in a similar manner described in Example VIII to give 5.2g of N-(2-mercaptopropionyl)S-aminocaproic acid.

(m.p. 78 79C) Infrared Spectrum Analysis Found: C. 49.26; H. 7.78; N. 6.42 Calcd.: C. 49.30; H. 7.82; N. 6.39

EXAMPLE X 820g (0.54 mol) of DL-phenylglycine was neutralized by dissolving it with 720 ml of 2N-NaOH aqueous solution. 931g (0.54 mol) of 2-bromopropionylchloride and 720 ml of 2N-NaOH aqueous solution was simultaneously added dropwise to the solution over one hour while stirring and ice-cooling. During the addition. the reaction system was maintained in a slightly alkaline condition. After completion of the addition, the ice bath was removed and the mixture was stirred for further 2 hours. Then. the mixture was acidified with hydrochloric acid to precipitate crystals. The crystals thus formed were recovered by filtration, washed with water and dried to give 147.3g of N-(2-bromopropionylphenyl)glycine. (m.p. 144 147C; Yield. 94.8%)

Infrared Spectrum 3200 cm '(NH); 1720 cm"(COOI-I); 1630 1 1 1g (0.39 mol) of N-(2-bromopropionylphenyl)glycine thus obtained was dissolved in 194 ml of 2N- NaOH aqueous solution. To the solution was added the filtrate. which had been prepared by neutralizing 58.9g (0.43 mol) of thiobenzoic acid with 214 ml of 2N- NaOH aqueous solution and filtering the neutralized solution. and followed by stirring overnight. On the next morning. the reaction mixture was acidified with hydrochloric acid to form oil. The oil was extracted with ethyl acetate. and the extract was dried over anhydrous sodium sulfate and distilled under reduced pressure to remove ethyl acetate to give 129g of crystals. These crystals were recovered by filtration and washed with benzene to give 838g of l\'( 2- benzoylmercaptopropionylphenyl)glycine. (m.p. 149 151C: Yield. 62.9%)

Infrared Spectrum cm. 1520 cm"(CONH-) To 34.3g (0.1 mol) of the S-benzoyl compound thus obtained was added 170 ml of aqueous ammonia thereby hydrolyzing the compound in the manner similar to that described in Example I to give 201g of N-( 2- mercaptopropionylphenyl)glycine.

(m.p. 136C; Yield. 84.1%)

Infrared Spectrum 3260 cm(--NH-): 1700 cm"(CO()H); 1635 cm, 1525 cm(CONH) Analysis Found: C. 55.17; H, 5.43; N. 5.62 Calcd: C. 55.23; H. 5.48; N. 5.86

We claim: 1. A compound represented by the formula SH R wherein when R is phenyl. benzyl or p-chlorophenyl. R is hydrogen and when R is methyl. R is phenyl or benzyl.

2. N-(2-mercaptophenylacetyl)glycine according to claim 1.

3. N-(2-mercapto-p-chloropheny1acetyl)glycine according to claim 1.

4. N-(2-mercaptopropionylphenyl)glycine according to claim I.

5. N-(2mercapto-3-phenylpropionyl)glycine cording to claim I.

Page 1 of 2 UNITED STATES PATENT AND TRADEMARK OFFICE PATENT NO.

DATED INV ENTOR(S) 3 CERTIFICATE OF CORRECTION July 29, 1975 ITARU MITA et a1 It is certified that error appears in the above-identified patent and that said Letters Patent are hereby corrected as shown below:

Abstract, line 1: rewrite "prophyaxis" as prophylaxis Column Column Column Column Column Column Column Column 1, line 12: replace "has" with had 2, line 15: after "mice" replace "was" with were 2 Table, last two lines:

after "propionyl", close replace "phenyl)glycine" parentheses A with phenylalanine 3, line 19: replace "salt" with salts 3 line 39: replace "acid" with aid 6, line 2: replace "zamide of" with zamide by 9, line 4: replace "was described" with as described 8, lines 42-43: replace "2benzoylmercaptopropionyl- B-alanine" with N- (2benzoylmercaptopropionyl B-alanine O Page 2 of 2 DATED July 29, 1975 Column 1, line 22: replace "consiting" with consisting UNITED STATES PATENT AND TRADEMARK OFFICE CERTIFICATE OF CORRECTION PATENT N0. 3,897,480

INVENTOR(S) I ITARU MITA et al It is certified that error appears in the above-identified patent and that said Letters Patent are hereby corrected as shown below:

Column 1, line 8: after "activities" and before "British",

insert an open parentheses and delete "and".

Column 6, lines 4-5: replace "2mercapto-p-chlorophenylacetylglycine" with N-(2mercapto-p-chlorophenylacetyl)glycine Column 10, line 20: replace "mercaptopropionylphenyl) glycine" with mercaptopropionyl)phenylglycine Column 10, line 50: replace "N- (2mercaptopropionylphenyl) glycine" with N- (2-mercaptopropiony1)phenylglycine Column 5, line 18: replace "mercapto-3-phenyl)glycine" with mercapto-3-phenylpropionyl)glycine a Signed and Scaled this sixteenth D ay 0 f March 1 9 76 [SEAL] L A ttes t.

RUTH C. MASON Arresting Officer C. MARSHALL DANN Commissioner uj'Parenis and Trademarks 

1. A COMPOUND REPRESENTED BY THE FORMULA
 2. N-(2-mercaptophenylacetyl)glycine according to claim
 1. 3. N-(2-mercapto-p-chlorophenylacetyl)glycine according to claim
 4. N-(2-mercaptopropionylphenyl)glycine according to claim
 1. 5. N-(2-mercapto-3-phenylpropionyl)glycine according to claim
 1. 6. N-(2-mercaptopropionyl)phenylalanine. 